Studies in Bcl10-(Ruland et al., 2001) and Malt1-deficient (Ruland et al., 2003; Ruefli-Brasse et al., 2003) mice revealed their essential role in the signaling cascade from the antigen receptors to the transcription factor NFκB. Moreover, chromosomal translocations leading to overexpression of Bcl10 or MALT1, or creating the constitutively active fusion protein API2-MALT1, all result in uncontrolled and stimulus-independent activation of NFκB (Zhang et al., 1999, Willis et al., 1999, Dierlamm et al., 1999, Sanchez-Izquierdo et al., 2003). Furthermore, this constant activation of NFκB is thought to play a role in the pathogenesis of certain MALT-lymphomas. The human MALT1 protein contains a caspase p20-like domain, preceded by a large pro-domain, consisting of a Death Domain (DD) and two Ig-like domains, and is therefore also referred to as the human paracaspase (Uren et al., 2000). As such, MALT1 is most similar to initiator caspases that possess longer pro-domains than effector caspases, whose pro-domain is very small. Proteolytic activation of the initiator caspases in the apoptosome most likely occurs via a conformational change of the active site attained through direct interaction with the apoptosome or via proximity-driven oligomerization facilitated by the apoptosome (Boatright et al., 2003; Bao and Shi, 2007). Although Uren et al. (2000) suggested that in API2-MALT1 fusions paracaspase activity may play a role, they could not prove proteolytic activity. Moreover, mutation of the catalytic activity didn't abolish all NFκB activity. Indeed, the proteolytic activity of MALT1 paracaspase is not generally accepted and so far, no proteolytic activity has been reported for MALT1/paracaspase.
T-cell receptor (TCR) engagement results in the formation of a highly ordered, membrane-associated complex called supra-molecular activation cluster (SMAC). Lipid rafts, which are sphingolipid- and cholesterol-rich microdomains in the cell membrane, are suspected to play an important role herein, as they migrate to the centre of the SMAC (cSMAC) to form larger clusters that function as signaling platforms. TCR stimulation and CD28 co-stimulation both activate a cascade of tyrosine phosphorylation that converges at lipid raft association and activation of PKCθ. Activated PKCθ serves different functions. On the one hand, it recruits TAK1 and the IKK complex to the periphery of the SMAC, resulting in TAK1-mediated phosphorylation of IKKβ (Shambharkar et al., 2007; Lee et al., 2005). On the other hand, PKCθ phosphorylates CARMA1 residing in the lipid rafts (Sommer et al., 2005; Matsumoto et al., 2005). This evokes a conformational change of CARMA1, allowing the recruitment of additional CARMA1 molecules (Sommer et al., 2005), BCL10 (Wang et al., 2002; Hara et al., 2004) and MALT1 (Che et al., 2004) to the lipid rafts in the cSMAC. Oligomerization of CARMA1 (Rawlings et al., 2006) triggers oligomerization of the BCL10-MALT1 complex, which in turn induces the oligomerization and activation of TRAF6 proteins via Lys63-linked auto-polyubiquitination (Sun et al., 2004). These polyubiquitin chains might assist CARMA1-dependent recruitment of the IKK complex in the cSMAC (Hara et al., 2004; Stilo et al., 2004) via the ubiquitin binding domains of IKKγ (Wu et al., 2006), which then culminates in full IKK activation via Lys63-linked polyubiquitination of IKKγ (Zhou et al., 2004). Activated IKK phosphorylates the NFκB inhibitory protein IκB (Sun et al., 2004), which marks it for Lys48-linked polyubiquitination and degradation by the proteasome (Chen, 2005), thereby releasing and activating NFκB. Important, the BCL10 protein appears to be a point of divergence in the NFκB pathway in T- and B-lymphocytes (Ruland et al., 2003). Whereas mature T-cells depend entirely on MALT1 to send information from BCL10 to NFκB, mature B-cells require MALT1 only for a BCL10-subprogram by specifically inducing c-Rel upon B-cell receptor stimulation, while BCL10 regulates both RelA and c-Rel activation (Ferch et al., 2007).
Recent work also identified BCL10 and MALT1 as central regulators of a specific signaling pathway that controls NFκB activation and proinflammatory cytokine production upon Fc epsilon RI ligation on mast cells. Mice deficient for either protein display severely impaired IgE-dependent late phase anaphylactic reactions (Klemm et al., 2006). Strong evidence suggesting that conserved BCL10-MALT1 complexes interact with different CARD scaffolds to connect various receptors in different cell types to NF-kB signaling has emerged more recently. The CARD10 (CARMA3)-Bcl10-Malt1 signalosome functions as a link between G protein-coupled receptor (GPCR) signaling and proinflammatory NF-kB activation. For example, CARMA3/Bcl10/MALT1 dependent NF-kB activation mediates angiotensin II-responsive inflammatory signaling in nonimmune cells (Allister-Lucas et al., 2007). The pathway is similar to the pathway described in lymphocytes, but CARMA1, which is found chiefly in lymphocytes is replaced by a family member with a wider tissue distribution profile, CARMA3. Similarly, BCL10 and MALT1 are critically required for NFκB induction in response to GPCR stimulation by lysophosphatidic acid (LPA) (Klemm et al., 2007). Further, Dectin-1 receptor-induced NFκB activation in dendritic cells depends on CARD9-BCL10-MALT1, indicating a role in responses to fungal infection. These results identify CARD-BCL10-MALT1 signalosomes as pivotal regulators that link not only innate and adaptive immune responses, but also GPCR signaling, to the canonical NF-kB pathway (reviewed by Wegener and Krappmann, 2007).
Studies in cell lines show that overexpressed MALT1 by itself does not activate NFκB, whereas co-expression with BCL10 results in a synergistic effect on BCL10-mediated NFκB activation (Lucas et al., 2001). Current hypothesis is that BCL10 facilitates MALT1 oligomerization and activation of associated TRAF6 proteins (Sun et al., 2004). However, it was also shown that mutation of the predicted catalytic cysteine (C464A) reduced the synergism with BCL10 (Lucas et al., 2001), though proteolytic activity has not been demonstrated so far, and the mechanism by which the reduced activity is caused was unknown till now.
Surprisingly we found that MALT1 shows proteolytic activity in vitro, with cysteine protease activity and that this activity can be detected using a tetrapeptide substrate. Co-expression of MALT1 and BCL10 or raft association of MALT1 induce its auto-proteolytic cleavage generating a 76 kDa C-terminal fragment that can activate NFκB signaling. Furthermore we demonstrate that MALT1 auto-proteolysis is involved in NFκB signaling. Moreover, we found that MALT1 proteolytic activity is activated upon TCR stimulation. More specifically, we demonstrated that MALT1 mediates the rapid proteolytic cleavage and inactivation of the NFκB inhibitor A20 (also known as TNFAIP3) upon TCR stimulation, resulting in increased TCR dependent IL-2 production.